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After approximately 16 hours of incubation with hormone, cells in each well were lysed using 100L mammalian protein extraction reagentAfter blocking, sections were stained with antibodies or phalloidin and imaged with a Leica DM4000B fluorescent microscopeAfter centrifugation, the halo containing PBMCs was carefully transferred into a clean centrifuge tube and washed twice with 7 ml of PBS
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After incubation, the two alternatives ended up merged and incubated for 20 minutes at RT to make it possible for complicated development to happenAfter incubation at room temperature for 30 min, liquid of each sample well was removed, washed and subsequently incubated with the color reaction TMB Chromogen in the dark for 15 min at room temperatureAfter incubation of lysates with an anti-Flag antibody, coimmunoprecipitated endogenous RBP-J proteins were being detected utilizing an anti-RBP-J antibody
After incubation with the appropriate peroxidase-conjugated secondary antibody (DAKO, Denmark), the immunoreactive bands were visualized by chemioluminescenceAfter pancreas disgregation, cells were stained with Dithizone to identify the Islets (marked by the arrow) as described in Materials and Methods sectionAfter prediction, the potential miRNA loci were examined carefully based on the distribution and numbers of small RNAs on the entire precursor regions
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